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Which Form of Dopamine Is the Substrate for the Human Dopamine Transporter: the Cationic or the Uncharged Species?

1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês

10.1074/jbc.274.8.4876

1083-351X

Janet L. Berfield, Lijuan C. Wang, Maarten E. A. Reith,

Receptor Mechanisms and Signaling

The question of which is the active form of dopamine for the neuronal dopamine transporter is addressed in HEK-293 cells expressing the human dopamine transporter. TheK m value for [3H]dopamine uptake fell sharply when the pH was increased from 6.0 to 7.4 and then changed less between pH 7.4 and 8.2. The K I for dopamine in inhibiting the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane binding displayed an identical pH dependence, suggesting that changes in uptake result from changes in dopamine recognition. Dopamine can exist in the anionic, neutral, cationic, or zwitterionic form, and the contribution of each form was calculated. The contribution of the anion is extremely low (≤0.1%), and its pH dependence differs radically from that of dopamine binding. The increase in the neutral form upon raising the pH can model the results only when the pK a1 (equilibrium neutral-charged) is set to a much lower value (6.8) than reported for dopamine in solution (8.86). The sum of cationic and zwitterionic dopamine concentrations remained constant over the entire pH range studied. These forms are the likely transporter substrates with pH-dependent changes occurring in their interaction with the transporter. The binding of dopamine, a hydroxylated phenylethylamine derivative, displays the same pH dependence as guanethidine, a heptamethyleniminoethyl- guanidine derivative fully protonated under our conditions. An ionizable residue in the transporter could be involved that does not interact with or impact the binding of bretylium, a quaternary ammonium phenylmethylamine derivative that is always positively charged and shows only a minor reduction in K I upon increasing pH. The question of which is the active form of dopamine for the neuronal dopamine transporter is addressed in HEK-293 cells expressing the human dopamine transporter. TheK m value for [3H]dopamine uptake fell sharply when the pH was increased from 6.0 to 7.4 and then changed less between pH 7.4 and 8.2. The K I for dopamine in inhibiting the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane binding displayed an identical pH dependence, suggesting that changes in uptake result from changes in dopamine recognition. Dopamine can exist in the anionic, neutral, cationic, or zwitterionic form, and the contribution of each form was calculated. The contribution of the anion is extremely low (≤0.1%), and its pH dependence differs radically from that of dopamine binding. The increase in the neutral form upon raising the pH can model the results only when the pK a1 (equilibrium neutral-charged) is set to a much lower value (6.8) than reported for dopamine in solution (8.86). The sum of cationic and zwitterionic dopamine concentrations remained constant over the entire pH range studied. These forms are the likely transporter substrates with pH-dependent changes occurring in their interaction with the transporter. The binding of dopamine, a hydroxylated phenylethylamine derivative, displays the same pH dependence as guanethidine, a heptamethyleniminoethyl- guanidine derivative fully protonated under our conditions. An ionizable residue in the transporter could be involved that does not interact with or impact the binding of bretylium, a quaternary ammonium phenylmethylamine derivative that is always positively charged and shows only a minor reduction in K I upon increasing pH. The dopamine (DA) 1The abbreviations DAdopamineDATDA transporterhDAThuman DATANOVAanalysis of varianceGBR 129351-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazineWIN 354282β-carbomethoxy-3β-(4-fluorophenyl)tropane 1The abbreviations DAdopamineDATDA transporterhDAThuman DATANOVAanalysis of varianceGBR 129351-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazineWIN 354282β-carbomethoxy-3β-(4-fluorophenyl)tropanetransporter (DAT) in neuronal plasma membranes clears DA from the (extra)synaptic space (1Zimanyi I. Lajtha A. Reith M.E.A. Naunyn-Schmiedeberg's Arch. Pharmacol. 1989; 340: 626-632Crossref PubMed Scopus (66) Google Scholar, 2Garris P.A. Ciolkowski E.L. Pastore P. Wightman R.M. J. Neurosci. 1994; 14: 6084-6093Crossref PubMed Google Scholar, 3McElvain J.S. Schenk J.O. Biochem. Pharmacol. 1992; 43: 2189-2199Crossref PubMed Scopus (139) Google Scholar) by an active uptake process with co-transport of Na+ and Cl− (for recent reviews see Refs. 4Povlock S. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 1-28Crossref Google Scholar and 5Rudnick G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Google Scholar) but probably not counter-transport of K+ (6Gu H.H. Rudnick G. Soc. Neurosci. Abstr. 1996; 22: 370Google Scholar). In calculating the overall stoichiometry of the neuronal DA uptake process as 2:1:1 for Na+:Cl−:DA, the authors have combined the evidence for co-transport of two Na+ ions and one Cl− ion per DA molecule with the assumption that the cationic form of DA is the substrate for uptake (7Krueger B.K. J. Neurochem. 1990; 55: 260-267Crossref PubMed Scopus (127) Google Scholar, 8Gu H. Wall S.C. Rudnick G. J. Biol. Chem. 1994; 269: 7124-7130Abstract Full Text PDF PubMed Google Scholar, 9Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar). DA has an amino group that can accept a proton and a phenolic hydroxyl group that can donate a proton; the second phenolic group has a pK avalue greater than 12. Therefore, except at extremely basic pH values where both hydroxyl groups can be dissociated, DA can exist as a cation (+H3NDOH, with D for DA skeleton), a zwitterion (+H3NDO−), a neutral form (H2NDOH), or an anion (H2NDO−). With pK a1 and pK a2 values of 8.86 and 10.5, respectively (10Armstrong J. Barlow R.B. Br. J. Pharmacol. 1976; 57: 501-516Crossref PubMed Scopus (70) Google Scholar, 11Lewis G.P. Br. J. Pharmacol. Chemother. 1954; 9: 488-493Crossref PubMed Scopus (66) Google Scholar, 12Mack F. Bönisch H. Naunyn-Schmiedeberg's Arch. Pharmacol. 1979; 310: 1-9Crossref PubMed Scopus (147) Google Scholar) (see "Experimental Procedures"), it can be calculated that at physiological pH, DA exists mostly as a cation, which has prompted the assumption that this is the active form for transport (7Krueger B.K. J. Neurochem. 1990; 55: 260-267Crossref PubMed Scopus (127) Google Scholar, 8Gu H. Wall S.C. Rudnick G. J. Biol. Chem. 1994; 269: 7124-7130Abstract Full Text PDF PubMed Google Scholar, 9Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar). Indeed, in the analogous cases of neuronal uptake of serotonin and norepinephrine, strong evidence for translocation of the cationic form has been advanced (13Rudnick G. Kirk K.L. Fishkes H. Schuldiner S. J. Biol. Chem. 1989; 264: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 14Gu H.H. Wall S. Rudnick G. J. Biol. Chem. 1996; 271: 6911-6916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Indirect evidence in favor of the cation also being the active form for DA uptake comes from site-directed mutagenesis studies (15Kitayama S. Shimada S. Xu H. Markham L. Donovan D.M. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar) and molecular modeling (16Edvardsen O. Dahl S.G. Mol. Brain Res. 1994; 27: 265-274Crossref PubMed Scopus (47) Google Scholar) implicating an interaction between the protonated amine group of DA and Asp-79 of the DAT. The question of the active monoamine form for uptake by the vesicular monoamine transporter was given a great deal of attention more than a decade ago, with mixed conclusions. A case has been made for the cationic (17Njus D. Kelley P.M. Harnadek G.J. Biochim. Biophys. Acta. 1986; 853: 237-265Crossref PubMed Scopus (172) Google Scholar, 18Darchen F. Scherman D. Desnos C. Henry J.P. Biochem. Pharmacol. 1988; 37: 4381-4387Crossref PubMed Scopus (32) Google Scholar) as well as the uncharged form (for review see Ref. 19Johnson R.G.J. Physiol. Rev. 1988; 68: 232-307Crossref PubMed Scopus (265) Google Scholar), and to our knowledge no new information on this issue is available.In the present study, the question of the active form of DA for the neuronal DAT is addressed by monitoring for the human DAT (hDAT) cloned by Janowsky and colleagues (20Eshleman A.J. Henningsen R.A. Neve K.A. Janowsky A. Mol. Pharmacol. 1994; 45: 312-316PubMed Google Scholar, 21Eshleman A.J. Stewart E. Evenson A.K. Mason J.N. Blakely R.D. Janowsky A. Neve K.A. J. Neurochem. 1997; 69: 1459-1466Crossref PubMed Scopus (43) Google Scholar) and the pH dependence of (i) uptake and binding of DA, which can be in an ionized or neutral form, and (ii) binding of guanethidine, which is positively charged in the pH range studied (see below), and bretylium, which is always positively charged as a quaternary ammonium salt. Uptake was measured by monitoring the accumulation of [3H]DA, and binding was assessed indirectly through inhibition of high affinity binding of the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35428), which interacts with a domain on the DAT that overlaps with the DA domain (5Rudnick G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Google Scholar, 15Kitayama S. Shimada S. Xu H. Markham L. Donovan D.M. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar, 16Edvardsen O. Dahl S.G. Mol. Brain Res. 1994; 27: 265-274Crossref PubMed Scopus (47) Google Scholar, 22Xu C. Coffey L.L. Reith M.E.A. Biochem. Pharmacol. 1995; 49: 339-350Crossref PubMed Scopus (45) Google Scholar, 23Reith M.E. Xu C. Zhang L. Coffey L.L. Naunyn-Schmiedeberg's Arch. Pharmacol. 1996; 354: 295-304Crossref PubMed Scopus (22) Google Scholar, 24Reith M.E.A. de Costa B. Rice K.C. Jacobson A.E. Eur. J. Pharmacol. 1992; 227: 417-425Crossref PubMed Scopus (71) Google Scholar). By comparing DA uptake and binding, we can determine whether changes in uptake as a function of pH are solely determined by changes at the level of DA recognition, the first step in the DA translocation cycle. In contrast to DA, which is a dihydroxylated phenylethylamine, bretylium is a nonhydroxylated bromophenylmethylamine in which the amine is quaternary (25Maxwell R.A. Ferris R.M. Burcsu J.E. Paton D.M. The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines. Raven Press, New York1976: 95-153Google Scholar), and therefore the formation of an overall neutral species is not possible for this compound. Bretylium is usually employed for its inhibitory effect on norepinephrine release (see Ref. 26Masuda M. Kanai S. Miyasaka K. Am. J. Physiol. 1998; 274: G29-G34PubMed Google Scholar) and monoamine oxidase (27Molinoff P.B. Brimijoin S. Axelrod J. Biochem. J. 1971; 123: 32P-33PCrossref PubMed Google Scholar). In addition, it can be taken up into dopaminergic cells (28Kamal L.A. Arbilla S. Galzin A.M. Langer S.Z. J. Pharmacol. Exp. Ther. 1983; 227: 446-458PubMed Google Scholar) and interferes with DA uptake into striatal synaptosomes (29Kammerer R.C. Amiri B. Cho A.K. J. Med. Chem. 1979; 22: 352-355Crossref PubMed Scopus (25) Google Scholar). Guanethidine is a compound derived from guanidine. The latter is a strong base with a pK a of 12.5 (30Steinmetz P.R. Balko C. N. Engl. J. Med. 1973; 289: 141-146Crossref PubMed Scopus (23) Google Scholar) and is therefore predominantly in the positive guanidinium ion form over a wide pH range up to 11. Guanethidine has a pK a1 of 11.4 and a pK a2 of 8.3 (31Katzung B.G. Katzung B.G. Basic & Clinical Pharmacology. Appleton & Lange, Norwalk, CT1995: 1-8Google Scholar). The former pK avalue describes the ability of guanethidine, at the pH values between 6.0 and 8.2 studied here, to carry one proton at the guanidine residue, whereas the latter pK a value represents the ability of the guanido group to accept an additional proton as a function of pH (at pH 8.2 in ∼50% of the molecules). In a similar but not identical manner as bretylium, guanethidine is known to interfere with catecholaminergic transmission in mammalian (32Collins G.G. West G.B. Br. J. Pharmacol. 1968; 34: 514-522Crossref PubMed Scopus (21) Google Scholar) and invertebrate (33Silinsky E.M. Br. J. Pharmacol. 1974; 51: 367-371Crossref PubMed Scopus (6) Google Scholar) systems. Guanethidine can act as a substrate for the norepinephrine transporter (34Sachs C. Acta Physiol. Scand. Suppl. 1970; 341: 1-66PubMed Google Scholar) and has been reported to inhibit neuronal uptake of norepinephrine (25Maxwell R.A. Ferris R.M. Burcsu J.E. Paton D.M. The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines. Raven Press, New York1976: 95-153Google Scholar), but its affinity for the DAT, to our knowledge, has not been reported. The dopamine (DA) 1The abbreviations DAdopamineDATDA transporterhDAThuman DATANOVAanalysis of varianceGBR 129351-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazineWIN 354282β-carbomethoxy-3β-(4-fluorophenyl)tropane 1The abbreviations DAdopamineDATDA transporterhDAThuman DATANOVAanalysis of varianceGBR 129351-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazineWIN 354282β-carbomethoxy-3β-(4-fluorophenyl)tropanetransporter (DAT) in neuronal plasma membranes clears DA from the (extra)synaptic space (1Zimanyi I. Lajtha A. Reith M.E.A. Naunyn-Schmiedeberg's Arch. Pharmacol. 1989; 340: 626-632Crossref PubMed Scopus (66) Google Scholar, 2Garris P.A. Ciolkowski E.L. Pastore P. Wightman R.M. J. Neurosci. 1994; 14: 6084-6093Crossref PubMed Google Scholar, 3McElvain J.S. Schenk J.O. Biochem. Pharmacol. 1992; 43: 2189-2199Crossref PubMed Scopus (139) Google Scholar) by an active uptake process with co-transport of Na+ and Cl− (for recent reviews see Refs. 4Povlock S. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 1-28Crossref Google Scholar and 5Rudnick G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Google Scholar) but probably not counter-transport of K+ (6Gu H.H. Rudnick G. Soc. Neurosci. Abstr. 1996; 22: 370Google Scholar). In calculating the overall stoichiometry of the neuronal DA uptake process as 2:1:1 for Na+:Cl−:DA, the authors have combined the evidence for co-transport of two Na+ ions and one Cl− ion per DA molecule with the assumption that the cationic form of DA is the substrate for uptake (7Krueger B.K. J. Neurochem. 1990; 55: 260-267Crossref PubMed Scopus (127) Google Scholar, 8Gu H. Wall S.C. Rudnick G. J. Biol. Chem. 1994; 269: 7124-7130Abstract Full Text PDF PubMed Google Scholar, 9Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar). DA has an amino group that can accept a proton and a phenolic hydroxyl group that can donate a proton; the second phenolic group has a pK avalue greater than 12. Therefore, except at extremely basic pH values where both hydroxyl groups can be dissociated, DA can exist as a cation (+H3NDOH, with D for DA skeleton), a zwitterion (+H3NDO−), a neutral form (H2NDOH), or an anion (H2NDO−). With pK a1 and pK a2 values of 8.86 and 10.5, respectively (10Armstrong J. Barlow R.B. Br. J. Pharmacol. 1976; 57: 501-516Crossref PubMed Scopus (70) Google Scholar, 11Lewis G.P. Br. J. Pharmacol. Chemother. 1954; 9: 488-493Crossref PubMed Scopus (66) Google Scholar, 12Mack F. Bönisch H. Naunyn-Schmiedeberg's Arch. Pharmacol. 1979; 310: 1-9Crossref PubMed Scopus (147) Google Scholar) (see "Experimental Procedures"), it can be calculated that at physiological pH, DA exists mostly as a cation, which has prompted the assumption that this is the active form for transport (7Krueger B.K. J. Neurochem. 1990; 55: 260-267Crossref PubMed Scopus (127) Google Scholar, 8Gu H. Wall S.C. Rudnick G. J. Biol. Chem. 1994; 269: 7124-7130Abstract Full Text PDF PubMed Google Scholar, 9Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar). Indeed, in the analogous cases of neuronal uptake of serotonin and norepinephrine, strong evidence for translocation of the cationic form has been advanced (13Rudnick G. Kirk K.L. Fishkes H. Schuldiner S. J. Biol. Chem. 1989; 264: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 14Gu H.H. Wall S. Rudnick G. J. Biol. Chem. 1996; 271: 6911-6916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Indirect evidence in favor of the cation also being the active form for DA uptake comes from site-directed mutagenesis studies (15Kitayama S. Shimada S. Xu H. Markham L. Donovan D.M. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar) and molecular modeling (16Edvardsen O. Dahl S.G. Mol. Brain Res. 1994; 27: 265-274Crossref PubMed Scopus (47) Google Scholar) implicating an interaction between the protonated amine group of DA and Asp-79 of the DAT. The question of the active monoamine form for uptake by the vesicular monoamine transporter was given a great deal of attention more than a decade ago, with mixed conclusions. A case has been made for the cationic (17Njus D. Kelley P.M. Harnadek G.J. Biochim. Biophys. Acta. 1986; 853: 237-265Crossref PubMed Scopus (172) Google Scholar, 18Darchen F. Scherman D. Desnos C. Henry J.P. Biochem. Pharmacol. 1988; 37: 4381-4387Crossref PubMed Scopus (32) Google Scholar) as well as the uncharged form (for review see Ref. 19Johnson R.G.J. Physiol. Rev. 1988; 68: 232-307Crossref PubMed Scopus (265) Google Scholar), and to our knowledge no new information on this issue is available. dopamine DA transporter human DAT analysis of variance 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazine 2β-carbomethoxy-3β-(4-fluorophenyl)tropane dopamine DA transporter human DAT analysis of variance 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazine 2β-carbomethoxy-3β-(4-fluorophenyl)tropane In the present study, the question of the active form of DA for the neuronal DAT is addressed by monitoring for the human DAT (hDAT) cloned by Janowsky and colleagues (20Eshleman A.J. Henningsen R.A. Neve K.A. Janowsky A. Mol. Pharmacol. 1994; 45: 312-316PubMed Google Scholar, 21Eshleman A.J. Stewart E. Evenson A.K. Mason J.N. Blakely R.D. Janowsky A. Neve K.A. J. Neurochem. 1997; 69: 1459-1466Crossref PubMed Scopus (43) Google Scholar) and the pH dependence of (i) uptake and binding of DA, which can be in an ionized or neutral form, and (ii) binding of guanethidine, which is positively charged in the pH range studied (see below), and bretylium, which is always positively charged as a quaternary ammonium salt. Uptake was measured by monitoring the accumulation of [3H]DA, and binding was assessed indirectly through inhibition of high affinity binding of the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35428), which interacts with a domain on the DAT that overlaps with the DA domain (5Rudnick G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Google Scholar, 15Kitayama S. Shimada S. Xu H. Markham L. Donovan D.M. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar, 16Edvardsen O. Dahl S.G. Mol. Brain Res. 1994; 27: 265-274Crossref PubMed Scopus (47) Google Scholar, 22Xu C. Coffey L.L. Reith M.E.A. Biochem. Pharmacol. 1995; 49: 339-350Crossref PubMed Scopus (45) Google Scholar, 23Reith M.E. Xu C. Zhang L. Coffey L.L. Naunyn-Schmiedeberg's Arch. Pharmacol. 1996; 354: 295-304Crossref PubMed Scopus (22) Google Scholar, 24Reith M.E.A. de Costa B. Rice K.C. Jacobson A.E. Eur. J. Pharmacol. 1992; 227: 417-425Crossref PubMed Scopus (71) Google Scholar). By comparing DA uptake and binding, we can determine whether changes in uptake as a function of pH are solely determined by changes at the level of DA recognition, the first step in the DA translocation cycle. In contrast to DA, which is a dihydroxylated phenylethylamine, bretylium is a nonhydroxylated bromophenylmethylamine in which the amine is quaternary (25Maxwell R.A. Ferris R.M. Burcsu J.E. Paton D.M. The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines. Raven Press, New York1976: 95-153Google Scholar), and therefore the formation of an overall neutral species is not possible for this compound. Bretylium is usually employed for its inhibitory effect on norepinephrine release (see Ref. 26Masuda M. Kanai S. Miyasaka K. Am. J. Physiol. 1998; 274: G29-G34PubMed Google Scholar) and monoamine oxidase (27Molinoff P.B. Brimijoin S. Axelrod J. Biochem. J. 1971; 123: 32P-33PCrossref PubMed Google Scholar). In addition, it can be taken up into dopaminergic cells (28Kamal L.A. Arbilla S. Galzin A.M. Langer S.Z. J. Pharmacol. Exp. Ther. 1983; 227: 446-458PubMed Google Scholar) and interferes with DA uptake into striatal synaptosomes (29Kammerer R.C. Amiri B. Cho A.K. J. Med. Chem. 1979; 22: 352-355Crossref PubMed Scopus (25) Google Scholar). Guanethidine is a compound derived from guanidine. The latter is a strong base with a pK a of 12.5 (30Steinmetz P.R. Balko C. N. Engl. J. Med. 1973; 289: 141-146Crossref PubMed Scopus (23) Google Scholar) and is therefore predominantly in the positive guanidinium ion form over a wide pH range up to 11. Guanethidine has a pK a1 of 11.4 and a pK a2 of 8.3 (31Katzung B.G. Katzung B.G. Basic & Clinical Pharmacology. Appleton & Lange, Norwalk, CT1995: 1-8Google Scholar). The former pK avalue describes the ability of guanethidine, at the pH values between 6.0 and 8.2 studied here, to carry one proton at the guanidine residue, whereas the latter pK a value represents the ability of the guanido group to accept an additional proton as a function of pH (at pH 8.2 in ∼50% of the molecules). In a similar but not identical manner as bretylium, guanethidine is known to interfere with catecholaminergic transmission in mammalian (32Collins G.G. West G.B. Br. J. Pharmacol. 1968; 34: 514-522Crossref PubMed Scopus (21) Google Scholar) and invertebrate (33Silinsky E.M. Br. J. Pharmacol. 1974; 51: 367-371Crossref PubMed Scopus (6) Google Scholar) systems. Guanethidine can act as a substrate for the norepinephrine transporter (34Sachs C. Acta Physiol. Scand. Suppl. 1970; 341: 1-66PubMed Google Scholar) and has been reported to inhibit neuronal uptake of norepinephrine (25Maxwell R.A. Ferris R.M. Burcsu J.E. Paton D.M. The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines. Raven Press, New York1976: 95-153Google Scholar), but its affinity for the DAT, to our knowledge, has not been reported.